Pathogenicity mechanisms of Campylobacter jejuni and Campylobacter fetus [Elektronische Ressource] : characterization of pathogenicity factors and signaling in host cell invasion / von Małgorzata Krause-Gruszczyńska, Krause

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• Publié par: otto-von-guericke-universitat_magdeburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 148
• Langue: English
• Poids de l'ouvrage: 57 Mo

Extrait

PATHOGENICITY MECHANISMS OF CAMPYLOBACTER JEJUNI
AND CAMPYLOBACTER FETUS: CHARACTERIZATION OF
PATHOGENICITY FACTORS AND SIGNALING
IN HOST CELL INVASION


Dissertation
zur Erlangung des akademischen Grades


doctor rerum naturalium
(Dr. rer. nat.)

genehmigt durch die Fakultät für Naturwissenschaften
der Otto-von-Guericke-Universität Magdeburg



von M.Sc. Małgorzata Krause-Gruszczyńska/Krause
geb. am 05.10.1979 in Gdynia, Polen




Gutachter:
PD Dr. Steffen Backert
Prof. Dr. Christof R. Hauck



eingereicht am 12.11.2007
verteidigt am 07.04.2008


















„Das Schönste, was wir erleben können,
ist das Geheimnisvolle.
Es ist das Grundgefühl,
das an der Wiege von wahrer Kunst
und Wissenschaft steht.
Wer es nicht kennt
und sich nicht mehr wundern,
nicht mehr staunen kann,
der ist sozusagen tot
und sein Auge erloschen.“

Albert Einstein

„The most beautiful thing we can experience,
is the mysterious.
It is the source of all true art and all science.
He to whom this emotion is a stranger,
who can no longer pause to wonder
and stand rapt in awe,
is as good as dead: his eyes are closed.“

Albert Einstein
Acknowledgements


I would like to thank Prof. Wolfgang König for making my work at the Institute of Medical
Microbiology possible.

I am very grateful to my supervisor P.D. Dr. Steffen Backert for his constant help and advice
and for giving me the opportunity to work in his group.

I express my gratitude to all cooperation partners for providing materials used in this study.

I wish to acknowledge all my friends and co-workers at the Institute of Medical Microbiology,
especially: Dr. Steffi Gieseler, Dr. Terry Kwok, Franziska Layer, Dr. Monika Sakowicz-
Burkiewicz, Diana Schmidt, Nicole Tegtmeyer, Bastian Thaa, Ruth Wittelsberger and Dana
Zabler for the friendly atmosphere in the laboratory and continuous exchange of ideas.
Special thanks go to Sabine Brandt for her constant encouragement and support.

I sincerely thank my mother and sister, who have supported me trough these years just being
there and giving their help whenever needed.

Finally, my loving thanks belong to my husband Michał Gruszczyński for his love, support
and patience during this work.

My work was supported by NBL3-PhD scholarship (01ZZ0407) from Magdeburger
Forschungsverbund, which is gratefully acknowledged.



Małgorzata Krause-Gruszczyńska








Part of this thesis has been published in following scientific papers:

Krause-Gruszczyńska M, van Alphen LB, Oyarzabal OA, Alter T, Hänel I, Schliephake A,
König W, van Putten JP, Konkel ME & Backert S (2007) Expression patterns and role of the
CadF protein in Campylobacter jejuni and Campylobacter coli. FEMS Microbiol Lett 274: 9-
16.

Krause-Gruszczyńska M, Rohde M, Hartig R, Genth H, Schmidt G, Keo T, König W, Miller
WG, Konkel ME & Backert S (2007) Role of the small Rho GTPases Rac1 and Cdc42 in host
cell invasion of Campylobacter jejuni. Cell Microbiol 9: 2431-2444. I Table of contents
Table of contents........................................................................................................I
List of figures........................................................................................................... IV
List of tables ............................................................................................................ VI
Abbreviations ......................................................................................................... VII
1. Abstract..............................................................................................................1
2. Zusammenfassung............................................................................................2
3. Introduction .......................................................................................................4
3.1. Epidemiology of Campylobacter species ........................................................ 4
3.2. Clinical manifestations .................................................................................... 6
3.3. Pathogenicity and virulence factors of Campylobacter jejuni........................... 7
3.4. Host cell factors involved in Campylobacter jejuni invasion........................... 14
3.5. Host inflammatory responses........................................................................ 15
3.6. Campylobacter jejuni surface structures and their role in evasion of host
immune responses....................................................................................... 17
3.7. Pathogenicity and virulence factors of Campylobacter fetus ......................... 19
3.8. A model of Campylobacter jejuni pathogenesis ............................................ 20
3.9. Aim of the study............................................................................................ 22
4. Materials & Methods..................................................................................23
4.1. Campylobacter strains and growth conditions............................................... 23
4.2. Escherichia coli (E. coli) strains and growth conditions ................................. 25
4.3. Plasmids....................................................................................................... 25
4.4. Eukaryotic cells and cell culture conditions ................................................... 27
4.5. Infection of host cells with Campylobacter strains......................................... 28
4.6. Transfection experiments.............................................................................. 28
4.7. RNA interference (RNAi)............................................................................... 28
4.8. Gentamicin protection assay......................................................................... 29
4.9. Inhibitor and activator studies ....................................................................... 30
4.10. GTPase activation assays ............................................................................ 31
4.11. Immunoprecipitation ..................................................................................... 32
4.12. Determination of protein concentration ......................................................... 33
4.13. Generation of the CadF and SapA antibodies............................................... 33
4.14. SDS polyacrylamide gel electrophoresis (SDS-PAGE) and
immunoblotting............................................................................................. 33
4.15. Enzyme-linked immunosorbent assay (ELISA) ............................................. 35
4.16. Isolation of DNA............................................................................................ 36
4.16.1. Determination of DNA concentration and quality........................................... 37
4.17. Restriction, modification and purification of DNA .......................................... 37
4.17.1. Cleavage of DNA with restriction enzymes ................................................... 37
4.17.2. DNA Ligation ................................................................................................ 38
4.17.3. Agarose gel electrophoresis ......................................................................... 38
4.17.4. Purification of DNA fragments and extraction from agarose gel .................... 39
4.18. Polymerase chain reaction (PCR)................................................................. 39
4.18.1. PCR Primers................................................................................................. 40 IITable of contents
4.19. Preparation of E. coli chemically competent cells.......................................... 40
4.20. Transformation of chemically competent E. coli ............................................ 41
4.21. Overexpression and purification of C. fetus surface array protein SapA........ 41
4.22. In Vitro SapA phosphorylation assay with recombinant c-Src or c-Abl .......... 42
4.23. Preparation of SapA-coated latex beads....................................................... 42
4.24. Analysis of the binding and internalization of SapA-coated latex beads
into host cells ................................................................................................ 42
4.25. Cell attachment assay .................................................................................. 43
4.26. Immunofluorescence labeling, Fluorescence and Confocal Laser
Scanning Microscopy (CLSM)....................................................................... 43
4.27. Field Emission Scanning Electron Microscopy (FESEM) .............................. 44
4.28. Matrix-assisted laser desorption/ionization-mass spectrometry
(MA